全 文 ： 周玥 等/利用农业废弃物固态发酵裂褶菌 F17 产锰过氧化物酶的基质优化
Chinese Journal of Biotechnology
http://journals.im.ac.cn/cjbcn March 25, 2014, 30(3): 524−528
DOI: 10.13345/j.cjb.130354 ©2014 Chin J Biotech, All rights reserved

Received: July 15, 2013; Accepted: September 18, 2013
Supported by: National Natural Science Foundation of China (No. 31070109), Nature Science Research Program of the Education Office
of Anhui Province (No. KJ2010A027).
Corresponding author: Rong Jia. Tel: +86-551-63861297; E-mail: ahdxjiarong@163.com
国家自然科学基金 (No. 31070109)，安徽省教育厅重点科研项目基金 (No. KJ2010A027) 资助。
网络出版时间：2013-11-05 网络出版地址：http://www.cnki.net/kcms/detail/11.1998.Q.20131105.1013.003.html
524 生 物 工 程 学 报

利用农业废弃物固态发酵裂褶菌 F17产锰过氧化物
酶的基质优化
周玥，杨兵，杨阳，荚荣
安徽大学生命科学学院，安徽 合肥 230601
玥周 , 杨兵, 杨阳, 等. 利用农业废弃物固态发酵裂褶菌F17产锰过氧化物酶的基质优化. 生物工程学报, 2014, 30(3): 524−528.
Zhou Y, Yang B, Yang Y, et al. Optimization of manganese peroxidase production from Schizophyllum sp. F17 in solid-state
fermentation of agro-industrial residues. Chin J Biotech, 2014, 30(3): 524−528.
摘 要 : 锰过氧化物酶 (MnP) 在环保领域有着广阔的应用前景。目前，利用廉价基质生产 MnP，尤其是利用
工农业废弃物生产 MnP 的研究受到了国内外学者的广泛关注。本实验利用响应面方法从几种不同的农业废弃
物中筛选裂褶菌 F17 (Schizophyllum sp. F17) 产 MnP 的固态发酵基质。结果表明，以 0.52∶0.15∶0.33 的比例
组成的松木屑、稻草和黄豆粉的混合基质为发酵产 MnP 的最佳基质，发酵第 6 天 MnP 的活力最高，达到 11.18 U/g。
因此，利用农业废弃物固态发酵产锰过氧化物酶在减低酶的成本和环境污染物治理方面具有重要的意义。
关键词 : 锰过氧化物酶，固态发酵，农业废弃物，优化
Optimization of manganese peroxidase production from
Schizophyllum sp. F17 in solid-state fermentation of
agro-industrial residues
Yue Zhou, Bing Yang, Yang Yang, and Rong Jia
College of Life Science, Anhui University, Hefei 230601, Anhui, China
Abstract: Manganese peroxidase (MnP), a crucial enzyme in lignin degradation, has wide potential applications in
environmental protection. However, large-scale industrial application of this enzyme is limited due to several factors
primarily related to cost and availability. Special attention has been paid to the production of MnP from inexpensive
sources, such as lignocellulosic residues, using solid-state fermentation (SSF) systems. In the present study, a suitable SSF
medium for the production of MnP by Schizophyllum sp. F17 from agro-industrial residues has been optimized. The mixed
solid medium, comprising pine sawdust, rice straw, and soybean powder at a ratio of 0.52:0.15:0.33, conferred a maximum
enzyme activity of 11.18 U/g on the sixth day of SSF. The results show that the use of wastes such as pine sawdust and rice
straw makes the enzyme production more economical as well as helps solve environmental problems.
Keywords: manganese peroxidase (MnP), solid-state fermentation (SSF), agro-industrial residues, optimization
生物育种与工艺优化
周玥 等/利用农业废弃物固态发酵裂褶菌 F17 产锰过氧化物酶的基质优化
cjb@im.ac.cn
525

Manganese peroxidase (MnP) (EC1.11.1.13) is
an extracellular heme-containing glycoprotein
produced by various basidiomycetous fungi and it
can oxidize a variety of phenolic and non-phenolic
substances, including lignin as well as a range of
recalcitrate pollutants, such as chlorinated aromatic
compounds, heterocyclic aromatic hydrocarbons,
various dyes, and synthetic high polymers [1-2]. Due
to the important degradative potential of MnP, there
is a general interest in producing this enzyme
through fermentation processes [3-4].
However, the cost of enzyme production is a
challenge that could seriously limit the potential
broad application of MnP[4]. In recent years,
solid-state fermentation (SSF) has gained increasing
interest because several studies have shown that SSF
has the advantages of higher product yields, less
wastage of water, and simpler downstream
processing, when compared with submerged
fermentation (SmF)[4-6]. Currently, SSF is accepted
as the most economical process for ligninolytic
enzyme production by fungi, because it can produce
higher yield of enzyme using cheaper materials, such
as agro-industrial wastes. Hence, in the present
study, various lignocellulosic residues (rice hulls,
pine sawdust, wheat bran, rice straw, and wheat
straw) were screened as growth medium for the
production of MnP. The advantages of carrying out
SSF using these agricultural wastes include decrease
in the cost of enzyme production, reduction in the
quantity of solid waste, and increase in environment-
friendly management of agro-industrial wastes[3-4,7].
The white-rot fungus Schizophyllum sp. F17, a
local organism isolated by our lab, has been
previously shown to have the potential for dye
decolorization and the ability to produce MnP when
grown on agro-industrial residues[8-9]. Thus, the aim
of the present study was to determine a suitable SSF
mixed medium for MnP production by
Schizophyllum sp. F17 from lignocellulosic residues,
and optimize the ratio of the medium components by
response surface methods (RSM) to obtain high MnP
yield. After optimization, the production of MnP
was enhanced and maximum enzyme activity was
achieved on the sixth day of SSF.
1 Materials and methods
1.1 Strain, SSF substrate preparation,
inoculation, and culture conditions
Schizophyllum sp. F17 was isolated from a
decayed wood chip pile in the vicinity of Hefei, China.
The fungus was cultured on potato dextrose agar (PDA)
slants for one week at 28 . The grown mycelium mat, ℃
collected from several slants and washed with sterile
water, was homogenized using a Waring blender.
Subsequently, 500 mL flasks containing 200 mL of
liquid medium were inoculated with 20 mL of mycelial
suspension, and the flasks were incubated at 28 on a ℃
rotary shaker at 120 r/min for 3 d. The homogenized
mycelia obtained from the crushed mycelial pellets
were inoculated into 500 mL Erlenmeyer flasks
containing 20 g of SSF substrate designed by
MINITAB 15 software and 20 mL of water (the water
holding capacity was 1 mL/g (water per dry medium)),
and the flasks were incubated at 28 for 6 d.℃
1.2 Experimental designs and data analysis
RSM are widely applied in the optimization and
modeling of the fermentation process. They provide
important information regarding the optimum level
of each variable, along with its interactions with
other variables[3]. In the present study, several kinds
of lignocellulosic residues such as rice hulls, pine
sawdust, wheat bran, rice straw, and wheat straw
were screened using RSM to obtain the optimum
medium components influencing MnP production in
the SSF, and the important factors as well as their
optimal levels were evaluated. The experimental
plan was developed by adopting the Plackett-
Burman factorial design and the Extreme-Vertices
design with MINITAB software. SPSS (version 15.0)
was used for regression analysis of the experimental
data, and analysis of variance (ANOVA) was
employed to estimate the statistical parameters.
1.3 Enzyme assay
MnP activity was determined by the increase in
absorbance at 240 nm based on the oxidation of
Mn2+ to Mn3+ [8]. One unit (U) of MnP activity was
defined as the oxidization of 1 μmol of Mn2+ per
minute at 25 ℃, and the activity was expressed as U
per g dry residue (substrate plus mycelium) (U/g).
Experiments were performed in triplicate, and the
results were expressed as the mean values.
2 Results and discussion
2.1 Effect of medium components on MnP
production
Although MnP production from fungi is
influenced by many parameters such as species, type of
cultivation, culture medium, and time, the medium
components are the critical factors [10-12]. Table 1 shows
the chemical composition of several agricultural
residue employed in the present work. Rice hulls, pine
sawdust, wheat bran, rice straw, wheat straw, and
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soybean powder as substrates for the growth of
Schizophyllum sp. F17 and production of MnP were
investigated using Plackett-Burman design. Accordingly,
12 experiments were carried out (Table 2).
By applying multiple regression analysis, the
predicted MnP activity (MPA) was obtained from Eq. (1):
MPA=−0.155+0.195A+0.497B+0.033C+1.069D+
0.128E+0.109F.
where A: rice hulls; B: pine sawdust; C: wheat bran;
D: rice straw; E: wheat straw; and F: soybean powder.
The statistical significance of the variables in the
above-mentioned model (Eq. (1)) was examined by
t-test and P-test (Table 3). The P-values given in Table 3
show that pine sawdust and rice straw had greater
effects on MnP activity than the other factors. The
coefficient of determination (R2; 94.92%), which
indicates the reliability of the model, demonstrated that
the model was consistent with the experiment results.

Table 1 Chemical composition of some agricultural residue
used for MnP production
Agricultural
residue
Ligin
(%)
Cellulose
(%)
Hemicellulose
(%) References
Rice hulls 19 54 34 [13]
Pine sawdust 30 40 25 [14]
Wheat bran  10.7 22.3 [15]
Rice straw 18 32.1 24 [16]
Wheat straw 19.8 39.4 21.4 [15]

Table 2 Experimental design and results of Plackett-
Burman design experiment (MINITAB software)
Run
Rice
hulls
(g)
Pine
sawdust
(g)
Wheat
bran
(g)
Rice
Straw
(g)
Wheat
straw
(g)
Soybean
powder
(g)
MnP
Activity
(U/g)
1 0 5 0 0 0 3 1.87
2 0 5 5 0 5 0 2.73
3 5 5 0 5 0 0 8.58
4 5 0 5 0 0 0 1.21
5 0 0 0 0 0 0 0.00
6 0 0 0 5 5 3 5.66
7 5 5 5 0 5 3 4.12
8 5 5 0 5 5 0 9.35
9 0 5 5 5 0 3 9.67
10 5 0 5 5 0 3 5.62
11 0 0 5 5 5 0 6.02
12 5 0 0 0 5 3 2.91

Table 3 Estimated regression coefficients for Eq. (1)
Term Effect Coefficient T P
Constant 4.812 15.46 0.000
Rice hulls 0.973 0.487 1.56 0.179
Pine sawdust 2.483 1.242 3.99 0.010
Wheat bran 0.167 0.083 0.27 0.800
Rice straw 5.343 2.672 8.59 0.000
Wheat straw 0.640 0.320 1.03 0.351
Soybean powder 0.327 0.163 0.52 0.622
R2 = 94.92%.

Residual plot was used for further analysis of the
model. The residual normal probability figure (Fig. 1a)
showed that the residual distribution conformed to the
normal distribution, implying that the experiment
results were positively correlated with the predictions.
In Fig. 1b illustrating the residual distribution, the
activities of MnP were randomly distributed on both
sides of the zero value, indicating that the order of the
test had no effect on the results; thus, each value of the
results was independent and valid. On the other hand,
the most important medium components were tested by
Pareto analysis which is based on the notion that, for
many events, roughly 80% of the effects come from
20% of the causes. As shown in Fig. 1c, the Pareto
Chart indicated that pine sawdust and rice straw were
the most important factors influencing MnP activity,
which was noted to be consistent with the result of
P-test.


Fig. 1 Residual Plots for Eq.1 and pareto chart for the effect of
each variable. (a) The residual normal probability plot of Eq. 1,
the normal probability plot produced an approximately straight
line, demonstrating the points conformed to a normal distribution.
(b) The residual distribution plot of Eq. (1), the points were
randomly distributed in both sides of zero value, indicating each
value of the result was independent and valid. (c) Pareto chart for
the effects of the variables, A: rice hulls; B: pine sawdust; C:
wheat bran; D: rice straw; E: wheat straw; F: soybean powder; B
and D had a significant effect on MnP activity.
周玥 等/利用农业废弃物固态发酵裂褶菌 F17 产锰过氧化物酶的基质优化
cjb@im.ac.cn
527
2.2 Determination of the ratio of SSF medium
components
After determining the key factors affecting MnP
activity, the ratio of the medium components was
optimized using RSM to obtain high MnP yield. Pine
sawdust and rice straw were used as the main factors to
determine the ratio of SSF medium components. As
soybean powder is rich in nitrogen and mineral
elements, allowing efficient growth of fungal hyphae
in a shorter period of fermentation, it was also
considered as a medium component and examined by
RSM for high MnP yield. The Extreme-Vertices design
of mixture experiment was adopted to study the effect
of these nutrient substrates (pine sawdust, rice straw,
and soybean powder) on MnP fermentation. The
experimental matrix and results are given in Table 4.
By applying multiple regression analysis, a
second-order polynomial prediction equation was
derived as follows (Eq. (2)):
MPA=0.171A+0.107B+1.247C+0.151AB+0.028AC−
0.074BC.
where A: pine sawdust; B: rice straw; and C:
soybean powder.
In addition, t-test and P-test were also used to
examine the statistical significance of the factors in the
model (Eq. (2)). The results showed that the term AB
(pine sawdust×rice straw) in Eq. (2) was significant at
the 1% level, implying that the interaction between
pine sawdust and rice straw had a significant impact on
MnP activity, while the other two terms (AC and BC)
were not significant. Using ANOVA, an R2 value of
0.972 3 was obtained, which confirmed that the
equation was highly reliable.
Subsequently, residual plot was also used to
further examine the model. The MnP activities
obtained (Table 4) were positively correlated with
the predictions and each value of the results was
noted to be independent and valid according to the
residual plot (data not shown).
Furthermore, the effects of pine sawdust, rice
straw, and soybean powder on MnP activity were
also investigated using the response surface plot. Fig. 2
illustrates that the expected MnP activity was
10.68 U/g, and that the optimized ratio of pine
sawdust, rice straw, and soybean powder was
0.52:0.15:0.33. In addition, the desirability of the
experiment was 0.929, indicating that the optimized
ratio of the medium components can achieve the
anticipated target of the experiment.
Following the optimization of mixture medium
to produce MnP, the enzyme activity in the
optimized medium was recorded from the third day
on a daily basis, and the results showed that the peak
of MnP activity (11.18 U/g) appeared on the sixth
day of fermentation.
The maximum MnP yield achieved in the present
study was observed to be about 6.5 times higher than
that obtained through the SSF of eucalyptus residue
(1.7 U/g) by Arantes et al [17]. On the other hand,
Gassara et al[4] achieved a higher yield of MnP using
agro-industrial wastes different from those employed
in the present study. Nevertheless, the medium with
natural pH used in the current study is easily available
and economical. Also, some important fermentation
parameters including incubation temperature, inoculum
size, moisture content and particle size should be further
optimized in the next step for maximum yield of MnP
production. Thus, the above-mentioned results indicate
that agro-industrial materials such as pine sawdust and
rice straw are suitable feedstock for the production of
high-quality MnP by Schizophyllum sp. F17.

Table 4 Experimental design and results of extreme
vertex design experiment (MINITAB software)
Run
Pine
sawdust
(g)
Rice
straw
(g)
Soybean
powder
(g)
MnP
activity
(U/g)
1 0.00 10.00 5.00 3.74
2 10.63 3.13 1.25 9.36
3 10.00 0.00 5.00 9.28
4 15.00 0.00 0.00 2.44
5 0.00 15.00 0.00 1.89
6 8.13 3.13 3.75 11.23
7 3.13 10.63 1.25 6.55
8 7.50 7.50 0.00 10.78
9 12.50 0.00 2.50 5.76
10 5.00 5.00 5.00 9.48
11 0.00 12.50 2.50 2.06
12 6.25 6.25 2.50 9.39
13 3.13 8.13 3.75 8.77



Fig. 2 Response surface plot showing MnP activity with the
optimized medium. A maximum activity of MnP was achieved
from the mixed media which contained 7.75 g pine sawdust,
2.25 g rice straw and 5 g soybean powder. The desirability of
experiment was 0.929.
ISSN 1000-3061 CN 11-1998/Q Chin J Biotech March 25, 2014 Vol.30 No.3
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In this investigation, a comparison of the various
agricultural wastes on the production of MnP by
Schizophyllum sp. F17 revealed that the optimized ratio
combination of pine sawdust, rice straw, and soybean
powder has the greatest effect, with 3.6-fold and 4.9-fold
increases of enzyme activity, respectively, compared
with the culture grown on medium with only pine
sawdust or rice straw (Table 4). The major component of
pine sawdust and rice straw is cellulose, followed by
hemicellulose or lignin (Table 1). Cellulose and
hemicelluloses are macromolecules constructed from
different sugars; whereas lignin is an aromatic polymer
synthesized from phenylpropanoid precursors. The
composition and proportions of these compounds vary
between plants. Lignocellulosic residues are rich in
carbon content and other vital nutrients essential for
fungal growth. Pine sawdust is not only a significant
carbon source, but also an important inducer for
ligninolytic enzymes production by white-rot fungi. Rice
straw has a rich cellulosic composition and has been
used as the substrates to screen for novel fungus with
powerful delignification capability[18]. Soybean powder
utilized in this study is rich in nitrogen content, vitamins
and other nutrients for medium, and when added to pine
sawdust and rice straw, it improves the C-N sources,
presenting ideal conditions for fungal growth and
enzyme production. So, presumably, the production of
MnP activity may be dependent on the type of used
substrate, owing to the differences in chemical
compositions and structure, also, the combined C, N
sources of different kinds of agricultural wastes can
improve the production of ligninolytic enzymes.
3 Conclusion
The present study examined the feasibility of
MnP production from white-rot fungi by using
agro-industrial residues as the SSF medium. Solid-state
medium comprising pine sawdust, rice straw, and
soybean powder at a certain ratio was found to be
suitable for MnP production by Schizophyllum sp. F17.
The use of mixed agro-industrial residues as the SSF
medium for producing MnP considerably lowered the
production cost, making this enzyme a potential
candidate for industrial application.
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(本文责编 郝丽芳)